PROJECT SUMMARY Most bacteria in natural and clinical settings grow as surface-attached biofilms, which are bacterial communities that have self-assembled into an encased matrix of extracellular polymeric substances (EPS). To form these bacterial biofilm communities and infect host cells, an intercellular signaling process described as quorum sensing (QS) is very important. For the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus, QS regulates the expression of many genes important to biofilm initiation, EPS production, and virulence. While much has been learned about select factors that regulate biofilm formation in vitro and in animal models, the specific mechanisms by which multispecies groups of cells form biofilms are not understood. Furthermore, the therapeutic capabilities needed to treat the increasingly virulent infections of the coming decades will require deeper insight into multi-species interactions and their regulation of multiple gene expression profiles as represented over extended spatial and temporal scales during host cell infection. The correlated mass spectrometric and Raman chemical imaging approaches that have been developed by our combined research group circumvent the limitations imposed by previous technology which allowed examination of either a small number of cells or entire cell populations that had been removed from the conditions of interest. In contrast our correlated chemical imaging methods allow the determination and spatial mapping of individual bacterial species and their microbial products within a mixed bacterial community growing in situ on surfaces. One of our long-term goals is the design of detection and diagnostic strategies informed by an understanding of bacterial interactions and signature biomolecule production. We will continue our work toward this goal by conducting mono-culture and co-culture experiments using Pseudomonas aeruginosa and Staphylococcus aureus as model pathogens. Correlated chemical imaging will be used to define the onset and range of intercellular quorum sensing signaling in space and time. The biosecretome and interactions of specific species in co-culture biofilms will be characterized for both intra- and inter-species interactions. Also, an ex vivo lung assay will be created to study the spatial biofilm secretome. This research will exploit the ability to spatially map specific chemical products produced by these and other pathogenic bacteria thereby yielding deep and therapeutically informative insights into host colonization, infection, and virulence.